Staining and Detection

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Brandon French
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shipping or storage Environmentally friendly > non-mutagenic Pour and run a mini-gel in less than hour Immediate band visualization EZ-Vision staining of DNA Molecular Weight Standards.

The gel was documented with a Syngene G-BOX Chemi HR imaging system with a short pass filter (380-650 nm).

Increased sensitivity Reduced backgrounds Western Blot Stripping Buffers 3 different formulations for antibody stripping from Western Blots Vantage ReView Stripping Buffer High Stringency - No

1 Third Quarter 2008 Volume 24, Number 3 A Quarterly Newsletter Featuring Product Information Published By AMRESCO Inc. Additonal Products Staining and Detection Non-mutagenic alternatives to Ethidium Bromide RiboZol RNA Extraction Reagent Purify high yields of total RNA in under an hour with AMRESCO s single phase organic solution. Fluorescent SPRINT NEXT GEL A Instantaneous visualization of DNA bands on agarose gels Convenient > supplied in loading buffer for single step staining and loading Economical > no hazardous shipping or storage Environmentally friendly > non-mutagenic Pour and run a mini-gel in less than hour Immediate band visualization EZ-Vision staining of DNA Molecular Weight Standards. A 1 kb DNA ladder (Code: K UL) was mixed with EZ-Vision Three and resolved on a 1% Agarose I (Code: 0710) gel in TAE running buffer. Bands were visualized by exposure to UV light. The gel was documented with a Syngene G-BOX Chemi HR imaging system with a short pass filter ( nm). VisiGlo & VisiGlo Plus HRP Chemiluminescent Substrates for Western Blotting/ELISA Chemiluminescent substrates for detection of peroxidase-labeled antibodies on Western blots and in ELISA assays. Increased sensitivity Reduced backgrounds Western Blot Stripping Buffers 3 different formulations for antibody stripping from Western Blots Vantage ReView Stripping Buffer High Stringency - No Odor Gentle ReView Stripping Buffer Moderate Stringency - No Odor ReView Buffer Solution Original Formulation Ready-to-use cell viability stains No Mix - No Mess Simply add to cell suspensions and load onto hemocytometer for counting Quick-View Fluorescent Stain Live cells fluoresce green Dead cells fluoresce red Fluorescent micrograph of Quick-View stained cells. Trypan Blue Colorometric Stain Live cells exclude stain Dead cells stain dark blue Bright field micrograph of trypan blue stained cells The Investigator Newsletter is printed on recycled paper with soy based inks.

ISO 9001 Certified ISO 13485 Certified Detection & Labeling Cell Viability and Differentiation Stains Quick-View Fluorescent Viability Stain A ready-to-use staining reagent for easy discrimination of

Minimal set-up and clean-up required. Fast > Cells can be visualized immediately upon addition of stain. No fixation or washing needed.

2 ISO 9001 Certified ISO Certified Detection & Labeling Cell Viability and Differentiation Stains Quick-View Fluorescent Viability Stain A ready-to-use staining reagent for easy discrimination of live and dead mammalian cells Convenient > Provided as a pre-mixed solution in an easy-to-use dropper bottle. Simply mix stain with cells and load on hemocytometer for counting. Minimal set-up and clean-up required. Fast > Cells can be visualized immediately upon addition of stain. No fixation or washing needed. Versatile > Stains both suspended and adherent cells Safe > Supplied dropper bottle minimizes contact with reagents The two fluorescent dyes included in Quick-View, Acridine Orange and Ethidium Bromide, allow rapid and easy recognition of live and dead cells when visualized by fluorescence microscopy. Acridine Orange stains live cells green while Ethidium Bromide stains dead cells red-to-orange, depending on the filter employed in the microscope. The excitation and emission maxima for Quick-View are summarized below. Dye Excitation Maxima (nm) Emission Maxima (nm) Color/Viability Acridine Orange Green/Live cells Ethidium Bromide Red/Dead cells...quick and easy to set-up Karen McColl, Ph.D Department of Pharmacology Case Western Reserve University Cleveland, Ohio Quick-View staining of hybridoma cells. Cell concentration was adjusted to 1x10 6 cells/ ml and diluted 1:1 with Quick-View. A 20 μl aliquot of the cell suspension was loaded onto a hemocytometer and imaged through fluorescence microscopy. Trypan Blue Solution A ready-to-use 0.4% Trypan Blue solution for cell viability determination Convenient > Provided as a ready-to-use solution Fast > Stained cells are ready for counting within 5 minutes Trypan Blue is a widely used colorometric dye that distinquishes live cells from dead ones by a dye exclusion method. Live cells exclude the dye and remain clear and translucent in the microscopic field. In contrast, trypan blue penetrates membranes of dead cells and stains them a dark blue color. Trypan Blue staining of hybridoma cells. Cell concentration was adjusted to 1x10 6 cells/ml and diluted 1:1 with 0.4% Trypan Blue Solution. A 20 μl aliquot of the cell suspension was loaded onto a hemacytometer for counting and photography through a bright-field microscope. 2

3 Detection & Labeling Gram Staining Kit Procedure Gram Staining Kit Easy-to-use gram staining kit for the rapid discrimination of gram-negative and gram-positive bacteria The Gram stain differentiates bacteria into two primary groups based on the reactivity of their cell walls with a crystal violet-iodine complex. The AMRESCO Gram Stain Kit includes all the components required for the complete staining procedure. Convenient > All components are supplied as ready-to-use solutions in convenient dropper bottles Cost Effective > Each kit stains approximately 125 slides Gram-positive bacteria Thick cell wall with peptidoglycan content between 50-90% absorbs and retains the crystal violet-iodine complex throughout the staining procedure. Appears purple in bright-field illumination in the microscope Gram-negative bacteria Thin cell wall with peptidoglycan content under 10% that is insufficient to retain the crystal violet-iodine complex during the staining procedure Appears pink in bright-field illumination in the microscope Gram positive staining of S. aureus Gram negative staining of B. hypermegas MTT (Thiazolyl Blue) A sensitive colorometric substitute for radioisotopes in cell proliferation and cytotoxicity studies MTT, a yellow tetrazole dye, can be reduced to a water-insoluble purple formazan compound by mitochondrial reductase enzymes. Since reduction only occurs in living cells the quantitation of formazan can be equated to the number of viable cells in the population. MTT MTT Formazan Applications: Cell Viability Assays Dose Response Curves Cytotoxicity Assays Quick-View Fluorescent Viability Stain Sufficient to prepare 250 slides N600-5ML 5 ml $76.20 Trypan Blue, 0.4% Solution K ML 100 ml $11.20 MTT (Thiazolyl Blue), 3-(4,5-Dimethylthylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide Gram Stain Kit Includes: Gram Safranin, 125 ml Gram Iodine, 125 ml Gram Crystal Violet, 125 ml Gram Decolorizer Solution, 125 ml Contains sufficient reagents for 125 slides MG G G 500 mg 1 g 5 g $30.80 $42.70 $ K918-KIT 1 KIT $

Detection & Labeling ISO 9001 Certified ISO 13485 Certified Protein Detection SDS-PAGE Detection Ready-to-pour SDS-PAGE solutions containing a fluorescent stain for immediate band visualization Gel

4 Detection & Labeling ISO 9001 Certified ISO Certified Protein Detection SDS-PAGE Detection Ready-to-pour SDS-PAGE solutions containing a fluorescent stain for immediate band visualization Gel casting through band visualization in < hour Bands visible within 5 minutes of exposure to UV illumination. No staining or destaining needed Staining sensitivity comparable to Coomassie Blue Ideal for downstream applications such as Western blotting Western Blot Detection VisiGlo and VisiGlo Plus HRP Chemiluminescent Substrates High sensitivity substrates for Western blotting and ELISA assays VisiGlo HRP and VisiGlo PLUS HRP are non-isotopic, luminol-based chemiluminescent substrates for detection of peroxidase-labeled antibodies on Western blots and in ELISA assays. They offer increased sensitivity and reduced backgrounds over competitor substrates for immunodetection applications. A B C kda kda 97.4 kda 66.2 kda 40.0 kda Available in 2 polyacyrlamide concentrations 10% (separation range 10kDa-200kDa) 12.5% (separation range 3.5 kda-100 kda) A B C D 31.0 kda 26.6 kda 21.5/19.7 kda 14.4 kda Rapid visualization of protein bands on Fluorescent SPRINT NEXT GEL. Molecular weight markers and total E. coli lysate were resolved on a 12.5% Fluorescent SPRINT NEXT GEL run at 300 volts for 30 minute. Bands were visualized by exposure to UV light for 3 minutes. Lane A: AMRESCO Wide Range Protein Molecular Weight Marker (Code: K494). Lane B: Total E. coli lysate. Lane C: AMRESCO Mid/Low Range Protein Molecular Weight Marker (Code: J450). ProLUMA Fluorescent Protein Gel Dye Using precast gels? Casting gels from your own solutions? Visualize sharp intense bands in 25 minutes No washing, no destaining! Visible bands within 3-5 minutes of irradiation on a UV transilluminator Sensitivity similar to Coomassie Blue Extremely low background ProLUMA staining of bacterial lysates. Total E. Coli lystate protein was resolved on a 10% NEXT GEL (Code: M256) and stained with ProLUMA for 20 minutes. Bands were visualized by exposure to UV light for 5 minutes (Lanes 1 & 2). The gel was then stained with Coomassie Blue R250 for 3 hours according to standard procedures (Lanes 3 & 4). Lanes 1 & 3: AMRESCO Mid/ Low Range Protein Molecular Weight Marker (Code: J µl). Lanes 2 & 4: Total E Coli lysates, 50 µg. Fluorescent SPRINT NEXT GEL, 10% Solution* Includes: NEXT GEL Running Buffers, 20 X Separation Range: 10 kda-200 kda Fluorescent SPRINT NEXT GEL, 12.5% Solution* Includes: NEXT GEL Running Buffers, 20 X Separation Range: 3.5 kda-100 kda 4 M317-KIT-100ML M317-KIT-500ML M318-KIT-100ML M318-KIT-500ML 100 ml 500 ml 100 ml 500 ml *Each 7.5 ml of NEXT GEL solution will prepare a 10 cm x10 cm by 0.75 mm mini-gel. NEXT GEL products are patent pending. ProLUMA Fluorescent Protein Gel Dye, 20X Solution VisiGlo HRP Chemiluminescent Substrate Kit Includes: VisiGlow HRP Chemiluminescent Substrate A VisiGlow HRP Chemiluminescent Substrate B VisiGlo Plus HRP Chemiluminescent Substrate Kit Includes: VisiGlow Plus HRP Chemiluminescent Substrate A, 120 ml VisiGlow Plus HRP Chemiluminescent Substrate B, 120 ml Comparison of VisiGlo and ECL chemiluminescent substrate sensitivity. Serial dilutions of Mouse IgG (1 ng 31 pg) were resolved by SDS-PAGE and transferred to PVDF membranes. Antigen was detected with HRP-labeled anti-mouse antibody. Film was exposed for 10 minutes. A) VisiGLO HRP; B) ECL Plus; C) VisiGLO PLUS HRP; D) ECL Advance. Feature VisiGlo HRP VisiGlo PLUS HRP Detection Limit Picogram Femtogram Working Solution Mix 2 components Mix 2 components Emission Duration 1-2 hours 4-8 hours Stability >1 year at 4ºC >1 year at 4ºC Recommended Membranes Nitrocellulose and PVDF M ML M308-10ML N218-KIT N218-S-KIT N218-Sample-KIT Nitrocellulose and PVDF Detection Method Film/Imager Film/Imager 125 ml 10 ml 120 ml (each substrate) 60 ml (each substrate) 5 ml (each substrate) $46.50 $ $46.50 $ $ $21.20 $ $72.40 $15.90 N219-KIT 1 Kit $420.00

5 Detection & Labeling Western Blot Stripping Buffers AMRESCO now offers 3 stripping buffer formulations for antibody removal from Western blots visualized with chemiluminescent substrates. Vantage ReView Stripping Buffer An odor-free, high stringency formulation for stripping tightly bound antibodies from Western blots Remove high-affinity antibodies with minimal loss of bound antigen Conserve scarce antigen and valuable antibodies Supplied as a ready-to-use solution βme Detection of IP3 Type 1 and Type 3 Receptors on a Single Western Blot 1 Type 1 3 Type Actin 72 hr 48 hr 40 hr 32 hr 24 hr 16 hr 8 hr 4 hr 0 hr 24 hr Veh Differential expression of IP3 Type 1 and Type 3 receptors in 1 µm Dexamethasone treated WEHI 7.2 mouse T cell lymphoma line. Courtesy of Dr. Karen McColl, Case Western Reserve University, Cleveland, Ohio. A single Western blot was probed with antibodies to the IP3 receptor Type I and then stripped for 65 minutes with Vantage ReView buffer. After removal of primary and secondary antibodies, the blot was reprobed with IP3 receptor Type 3 antibody. Successful removal of Type 1 antibody is demonstrated by the differential expression of Type 1 and Type 3 antibodies on the same blot. Gentle ReView Stripping Buffer An odor-free formulation for removal of most antibodies from Western blots Minimize loss or destruction of valuable antigen from membrane Ideal for multiple rounds of stripping and reprobing on a single membrane Supplied as a ready-to-use solution May be used at room temperature without a hood! βme I tried Gentle [ReView ] for 30 mins at 37º C with shaking, and I go ot 100% removal of both primary and secondary [antibodies].... I would love to use this for my regular projects. ReView Buffer Solution Original high stringency formulation Competitively priced Convenient: simply add β-mercaptoethanol to supplied solution Gentle ReView Stripping Buffer N552-1L 1L $75.60 ReView Buffer Solution N166-1L 1L $41.90 Vantage ReView Stripping Buffer N ML 500 ml $97.00 Samarjit Das, Ph.D. Department of Cardiac Pathology Johns Hopkins University School of Medicine, Baltimore, MD Technical Tip Analyzing antibody removal: 1. Wash blot with blocking buffer several times. 2. Incubate blot with secondary antibody. 3. Re-expose blot to chemiluminescent substrate. If no signal is detected, the primary antibody was effectively removed. 4. If a signal is detected, the blot must be stripped further. An increase in time or temperature can usually improve the effectiveness of the stripping process. 5

Detection & Labeling ISO 9001 Certified ISO 13485 Certified DNA Band Detection in Agarose Gels EZ-Vision DNA Dye as Loading Buffer A non-mutagenic alternative to Ethidium Bromide A non-mutagenic DNA

6 Detection & Labeling ISO 9001 Certified ISO Certified DNA Band Detection in Agarose Gels EZ-Vision DNA Dye as Loading Buffer A non-mutagenic alternative to Ethidium Bromide A non-mutagenic DNA dye supplied in 6X agarose gel loading buffer that provides instant band visualization upon illumination with UV light. EZ-Vision Protocol Mix 1 part EZ-Vision with 5 parts sample Safe > Results from an independent testing laboratory demonstrate that EZ-Vision is non-mutagenic in the Ames test Economical > No hazardous shipping or storage charges Convenient > Simply mix with sample and load on gel Time-saving > Immediately visualize bands on an UV transilluminator Performance > Extremely low background fluorescence Environmentally friendly > No mutagenic chemicals The proprietary dye included in EZ-Vision loading buffer binds to double-stranded DNA when mixed with the sample and co-migrates with it during electrophoresis. Gel bands are visible upon exposure to irradiation on UV transilluminators. EZ-Vision stained DNA is compatible with most downstream applications including cloning and PCR procedures. Load and run gel Available in 2 formulations EZ-Vision Three includes 3 tracking dyes three EZ-Vision One includes 1 tracking dye one 1 2 3,000 bp 2,000 bp 1,500 bp Place gel on UV transilluminator for instant band visualization Contains three tracking dyes for approximating fragment length Tracking dye mobility in 1% agarose gels: Magenta: ~10 bp Dark blue: ~600 bp Light Blue: ~4000 bp Try both formulations with the convenient sample kit EZ-Vision Sample Kit Each kit contains a 1 ml sample of each EZ-Vision One and EZ-Vision Three Contains a single, fast running tracking dye for a clear view of the entire gel Tracking dye mobility in 1% agarose gels: Magenta: ~10 bp 6 Staining of a 100 bp ladder with EZ-Vision Three and EZ-Vision ONE. Samples of an 100 bp Ladder (Code: K UL) were mixed with either EZ-Vision Three or EZ-Vision ONE and resolved on a TAE/2% Agarose I gel (Code: 0710). Lane 1: EZ-Vision Three stained ladder. Lane 2: EZ-Vision ONE stained ladder. Bands were visualized by exposure to UV light and the gel was documented with a Syngene G:BOX Chemi HR imaging system with a short pass filter ( nm). EZ-Vision ONE, DNA Dye as Loading Buffer, 6X Includes 1 tracking 10 bp on 1% agarose EZ-Vision Three, DNA Dye as Loading Buffer, 6X Includes 3 tracking 10 bp, 600 bp, 4000 bp 1,000 bp 900 bp 800 bp 700 bp 600 bp 500 bp 400 bp 300 bp 200 bp 100 bp EZ-Vision Sample Kit, DNA Dye as Loading Buffer, 6X Includes: EZ-Vision Three DNA Dye as Loading Buffer, 6X, 1 ml EZ-Vision ONE, DNA Dye as Loading Buffer, 6X, 1 ml N472-KIT N472-Q-0.5ML N313-KIT N313-Q-SAMPLE 5 x 1 ml 0.5 ml 5 x 1 ml 0.3 ml $ $19.10 $ $26.50 N473-2PK 2 x 1 ml $45.00

7 Detection & Labeling RNA Extraction Reagent Introducing AMRESCO s ready-to-use RNA extraction reagent RiboZol RNA Extraction Reagent Purify high yields of total RNA from a variety of cell and tissue types High Yields > Obtain total RNA from even the most difficult of cells and tissues types Rapid Procedure > Extraction takes less than 1 hour Reproducible > Consistent results with excellent recoveries Versatile > Compatible with a variety of applications including Northern analysis, dot blots, cloning, in vitro translation, polya+ selection and RT-PCR RiboZol Extraction Protocol Homogenization Add Chloroform Sample RNA Extraction Reagent Centrifuge RiboZol is a ready-to-use single phase organic solution for the isolation of total RNA from even the most difficult cell and tissue types. The sample is disrupted directly into RiboZol to inhibit RNase activity and prevent RNA degradation during purification. RNA obtained by RiboZol purification procedures is compatible with a variety of of downstream applications. Phase Separation RNA DNA Protein Transfer aqueous phase to a new tube Purity of RiboZol Total RNA Isolated isolated Total in RiboZol RNA Compared Compared to to Total RNA Total Isolated RNA Isolated with a with Leading an Alternative Competitor s Reagent Product 2.00 RNA Total RNA was was isolated from various tissues tissues and cells and cells with AMRESCO s RiboZol TM RNA Extraction with AMRESCO s Reagent RiboZol (magenta RNA Extraction bars) Reagent or 1.50 a (magenta) leading or competitor s with an alternative RNA reagent Extraction (purple). Reagent Purity was (purple assessed by bars). A Purity of RNA was assessed by A 260 /A 260 /A 280 ratio. 280 ratio. A 260 /A Kidney Brain K562 Cells Alternative Competitor Extraction Reagent Reagent AMRESCO RiboZol Reduced Genomic DNA Contamination in Ribozol Extracted RNA RNA Precipitation & Wash RNA Solubilization Add Isopropanol Centrifuge Remove supernatant RNA Pellet Wash Pellet Solubilize the RNA Pellet RiboZol RNA Extraction Reagent N ML N ML 100 ml 200 ml $95.00 $ Low Genomic DNA Contamination in RiboZol Extracted RNA. Courtesy of Dr. June Yun, North East Ohio University College of Medicine A Cos-7 cell culture was divided into 2 portions for RNA extraction with either RiboZol or with an alternative reagent. Lanes 2-4: RiboZol extracted RNA. Lanes 5-7: RNA extracted with an alternative reagent. 7

PRESORTED STANDARD U.S. POSTAGE PAID Cleveland, Ohio Permit No. 3630 AMRESCO Inc. 6681 Cochran Rd.

com Customer Service: cust-serv@amresco-inc.com Domestic Sales & Tech Support: dom-sales@amresco-inc.com Visit our Website at: www.amresco-inc.com The InvestiGator is published quarterly by the Marketing Department at AMRESCO Inc.

com Add a name to our mailing list by email, phone or fax: TEL 800-448-4442 FAX 440-349-0235 MOVING? Send us your new address to receive the latest news from the AMRESCO InvestiGator.

We supply custom packaging & formulations, which can meet your individual specifications.

8 PRESORTED STANDARD U.S. POSTAGE PAID Cleveland, Ohio Permit No AMRESCO Inc Cochran Rd. Solon, OH USA PHONE: (Toll Free) (Local) FAX: To Place Orders: General Information: Customer Service: Domestic Sales & Tech Support: Visit our Website at: The InvestiGator is published quarterly by the Marketing Department at AMRESCO Inc. Please direct all requests, product information inquiries, submissions, and/or comments to: AMRESCO Marketing Dept. Add a name to our mailing list by , phone or fax: TEL FAX MOVING? Send us your new address to receive the latest news from the AMRESCO InvestiGator. BUlk QUANtitiES & CUStOM CAPABilitiES AMRESCO can supply bulk quantities, sizes or a variety of grades of material. We supply custom packaging & formulations, which can meet your individual specifications. Specializing in production scale quantities and custom manufactured products, AMRESCO can support your individual company needs from pilot programs up to and through full-scale production requirements. We can produce materials with the exact composition, properties, and purity to fulfill your needs. Contact Our Bulk/OEM Sales Division for Additional information at: AMRESCO s Catalog Obtain your copy by ing us at: info@amresco-inc.com Please include your complete mailing address. ISO & ISO Certified Life Science Research Products Exhibitor Tradeshow Society for Neuroscience Washington D.C. Nov Copyright by AMRESCO Inc. All rights reserved. AMRESCO is a registered trademark of AMRESCO Inc. Al LiGator name and all Al LiGator graphic images are trademarks of AMRESCO Inc. EZ-Vision, Quick-View, RiboZol, NEXT GEL, VisiGlo, VisiGlo Plus, ReView, ProLUMA and RiboZol are trademarks of AMRESCO Inc. Pricing subject to change at AMRESCO s discretion.

EZ Vision DNA Dye as Loading Buffer

EZ Vision DNA Dye as Loading Buffer Code Description Size N472-1ML N472-KIT N650-1ML N650-KIT N313-1ML N313-KIT N473-2PK N473-3PK EZ Vision One DNA Dye as Loading Buffer, 6X EZ Vision Two, DNA Dye as Loading